Although obesity is associated with multiple metabolic risk factors for cardiovascular disease, including insulin resistance, diabetes, and dyslipidemia, many obese persons do not have obvious metabolic abnormalities. It is estimated that at least one-fourth of obese adults are metabolically normal, usually defined by some measure of insulin sensitivity or having d1 cardiometabolic abnormality. It is not known why weight gain and body fat accumulation causes metabolic abnormalities in some obese persons but not in others. Studies conducted during the last cycle of this grant found that excessive intrahepatic triglyceride (IHTG) content is a robust marker of obese persons who have metabolic dysfunction (insulin resistance in liver, muscle and adipose tissue and increased VLDL-triglyceride [VLDL-TG] secretion rate), independent of body mass index (BMI), percent body fat, and visceral fat mass. Conversely, obese persons who have normal IHTG content appear to be resistant to developing obesity-related metabolic complications. The mechanisms responsible for the accumulation of ectopic fat and the development of metabolic disease in some, but not all, obese persons are not known. Therefore, the overall goals of this proposal are: 1) to provide a better understanding of the metabolic mechanisms responsible for the accumulation of excessive ectopic fat (intrahepatic triglyceride [IHTG] and intramuscular triglyceride [IMTG]) and metabolic dysfunction in obese persons, and 2) to determine the specific metabolic adaptations that prevent the adverse metabolic effects of weight gain in obese persons who have normal IHTG content compared with those who have nonalcoholic fatty liver disease (NAFLD). A combination of in vivo and ex vivo methods will be used to determine the effect of overfeeding an additional 1000 kcal/d for ~8-10 wks, until a ~5% weight gain is achieved, on body triglyceride distribution and adipose tissue remodeling, multi-organ insulin sensitivity, and the mechanisms responsible for these changes in obese subjects who have either normal IHTG (metabolically- normal obesity) or high IHTG (metabolically-abnormal obesity) content. We hypothesize that overfeeding- induced abnormalities in lipid metabolism (increased adipose tissue lipolytic activity, increased hepatic and muscle tissue FFA uptake, impaired hepatic fatty acid oxidation and increased hepatic de novo lipogenesis) simultaneously cause ectopic fat accumulation, adipose tissue remodeling, inflammation and multi-organ insulin resistance. These studies will lay the groundwork for understanding why some obese persons are resistant, while others are prone, to developing obesity-related metabolic disease, which could have considerable future research and clinical implications. PUBLIC HEALTH RELEVANCE: Although obesity is associated with multiple metabolic risk factors for cardiovascular disease, including insulin resistance, diabetes, and dyslipidemia, many obese persons do not have obvious metabolic abnormalities. It is not known why weight gain and body fat accumulation causes metabolic abnormalities in some obese persons but not in others. We have found that excessive intrahepatic triglyceride (IHTG) content is a robust marker of obese persons who have metabolic dysfunction (insulin resistance in liver, muscle and adipose tissue and increased VLDL-triglyceride secretion rate), independent of body mass index, percent body fat, and visceral fat mass. Conversely, obese persons who have normal IHTG content appear to be resistant to developing obesity-related metabolic complications. The mechanisms responsible for the accumulation of ectopic fat and the development of metabolic disease in some, but not all, obese persons are not known. Therefore, the overall goals of this proposal are: 1) to provide a better understanding of the mechanisms responsible for the accumulation of excessive IHTG and ectopic fat associated with metabolic dysfunction in obese persons, and 2) to determine the specific metabolic adaptations that prevent the adverse metabolic effects of overfeeding and weight gain in obese persons who have normal IHTG compared with those who have nonalcoholic fatty liver disease. These studies will lay the groundwork for understanding why some obese persons are resistant, while others are prone, to developing obesity-related metabolic disease, which could have considerable future research and clinical implications.